WO2024131012A1 - 一种分析海洋刺胞生物中微塑料含量与分布的方法 - Google Patents

一种分析海洋刺胞生物中微塑料含量与分布的方法 Download PDF

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WO2024131012A1
WO2024131012A1 PCT/CN2023/103529 CN2023103529W WO2024131012A1 WO 2024131012 A1 WO2024131012 A1 WO 2024131012A1 CN 2023103529 W CN2023103529 W CN 2023103529W WO 2024131012 A1 WO2024131012 A1 WO 2024131012A1
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microplastics
microplastic
content
fluorescent
marine
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王伟
胡继荣
鲍茜
叶飞扬
叶庆富
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Zhejiang University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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  • the present invention belongs to the field of environmental pollutant detection and relates to a method for analyzing the content (enrichment) and distribution of microplastics in marine cnidarians, especially living jellyfish. It is a method for locating and quantitatively detecting microplastic particles based on fluorescent tracing technology.
  • microplastic particles Due to the widespread use of plastic products, they can be decomposed into microplastic particles with a diameter of less than 5mm under the action of physical, chemical and biological factors. These microplastic particles are widely present in water bodies such as rivers and oceans. After being ingested by organisms, they show a variety of biological toxicity and pose a potential threat to aquatic organisms.
  • microplastics are difficult to distinguish from other similar environmental particles in the field of view, it is necessary to use Fourier transform infrared spectrometers, Raman characterization, etc. to qualitatively characterize the particles, which increases the workload and complexity of positioning and quantifying microplastics in organisms.
  • the data obtained by counting methods are subjective and have sampling errors, and it is impossible to accurately quantify the content of microplastics.
  • the present invention provides a method for analyzing the content and distribution of microplastics in marine cnidarians, comprising:
  • Exposure experiment Fluorescent microplastic particles of a specific diameter were selected and added to artificial seawater to form an exposure solution system, and marine cnidarians were selected as subjects to carry out exposure experiments.
  • Stereo fluorescence microscopy observation The living body or local tissue of the exposed subject is transferred to a stereo fluorescence microscope, and the distribution of microplastics in the whole subject or local tissue is observed using a stereo fluorescence microscope.
  • Sample fluorescence imaging and calculation of microplastic content The exposed subject's living body or local tissue is placed in the detection room of the small animal living imager, and the small animal living imager is used to measure the fluorescence intensity in the living body or local tissue of the subject, and the corresponding content is calculated according to the standard curve; wherein the fluorescent microplastic standard curve is obtained by drawing the following method:
  • Solutions containing different amounts of fluorescent microplastics are added dropwise into a culture dish and placed in the detection chamber of a small animal living imaging instrument. After setting specific excitation and emission wavelengths, the fluorescence intensity of each solution is measured, and a fluorescent microplastic standard curve is drawn based on the microplastic content and the corresponding fluorescence intensity.
  • test subjects are marine cnidarians, such as jellyfish or sea anemones.
  • the salinity of artificial seawater is 20 ⁇ -30 ⁇ ; the temperature of the exposure solution is 18-25°C; and the diameter of the microplastics is 100-1000nm.
  • a culture dish is used as a container to transfer the living body or local tissue of the exposed subject to a stereoscopic fluorescence microscope and a detection room of a small animal living imaging instrument.
  • the fluorescence microscope filter is GFP-B;
  • the exposure experiment specifically includes: placing the test object in an exposure solution system and culturing it for a period of time, then taking it out, washing it with artificial seawater and then testing it.
  • the exposure experiment also includes setting up a blank control group for sample fluorescence imaging and microscopy.
  • the background fluorescence value of the test object is deducted in the calculation of the plastic content, and the marine cnidarians in the blank control group are cultured in artificial seawater.
  • a small animal in vivo imaging device is used to perform fluorescence imaging on the living subject.
  • it also includes: freeze-drying the tested subjects and weighing the dry weight of the samples, and calculating the content of microplastics per unit mass of tissue based on the microplastic content obtained by fluorescence imaging of the samples.
  • freeze-drying time is 48 hours.
  • the present invention has the following advantages: the method of the present invention directly uses a stereoscopic fluorescence microscope and a small animal live imager to detect microplastics in marine cnidarians, without the need for processing processes such as digestion of biological tissues. It can not only accurately locate the distribution of microplastics in living marine cnidarians, but also greatly simplify the detection process, and effectively and accurately quantify the content of microplastics in marine cnidarians, which is of great significance for the monitoring and control of new environmental pollutants, and provides a method for studying the enrichment ability of microplastics in marine cnidarians.
  • FIG. 1 is a flow chart of the method of the present invention.
  • Figure 4 Standard curve of fluorescence intensity of fluorescent microplastics as a function of content.
  • Figure 5 In vivo fluorescent imaging of jellyfish exposed to seawater with different concentrations of fluorescent microplastics.
  • Figure 6 The content of fluorescent microplastics in jellyfish.
  • the present invention discloses a method for analyzing the content and distribution of microplastics in marine cnidarians, laying a foundation for the health risk assessment of microplastics.
  • the process of the method is shown in Figure 1, and comprises the following steps:
  • the method for drawing a standard curve of fluorescent microplastics is: adding solutions containing different contents of fluorescent microplastics to a culture dish, placing it in the detection room of a small animal live imaging instrument, setting a specific excitation wavelength and emission wavelength (based on the selected fluorescent marker), and then measuring the fluorescence intensity of each solution, and drawing a standard curve with the microplastic content and the corresponding fluorescence intensity;
  • the step (1) uses fluorescently labeled microplastic particles with a diameter of 20-1000 nm;
  • the subject used in step (1) is jellyfish, such as jellyfish mother, jellyfish, etc.;
  • a method for analyzing the content and distribution of microplastics in marine cnidarians comprises the following steps:
  • Jellyfish exposure experiment Polystyrene microspheres (PS-MP, diameter 1 ⁇ m) labeled with NBD-CL (excitation wavelength 488 nm, emission wavelength 518 nm) fluorescent dye were used as the test object and added to artificial seawater to prepare the exposure solution.
  • the test animals were healthy jellyfish larvae (umbrella diameter 2.5 ⁇ 0.5 cm), which were randomly divided into an exposure group and a control group.
  • the exposure group included three polystyrene microsphere concentration levels (0.1, 1, and 10 mg L -1 ), and the exposure period was 5 days.
  • the method of the present invention directly uses a stereoscopic fluorescence microscope and a small animal live imager to detect microplastics in marine cnidarians, without the need for processing procedures such as digestion of biological tissues. It can not only accurately locate the distribution of microplastics in living marine cnidarians, but also greatly simplify the detection process and effectively quantify the microplastic content in marine cnidarians, which is of great significance for the monitoring and control of new environmental pollutants.

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Abstract

一种分析海洋刺胞生物中微塑料含量与分布的方法，包括：海洋刺胞生物暴露实验；体式荧光显微镜观察；荧光微塑料标准曲线绘制；样本荧光成像与微塑料含量计算等；本方法能准确定位微塑料在活体海洋刺胞生物体内的分布，并有效对生物体内和局部组织中的微塑料含量进行准确定量，对新型环境污染物的监测与治理具有重要意义。

Description

一种分析海洋刺胞生物中微塑料含量与分布的方法 技术领域
本发明属于环境污染物检测领域，涉及了一种分析海洋刺胞生物尤其是水母活体中微塑料含量(富集)与分布的方法，是一种基于荧光示踪技术的微塑料颗粒定位定量检测方法。
背景技术
由于塑料制品的广泛使用，在物理、化学、生物等因素作用下，塑料制品可以分解成直径小于5mm的微塑料颗粒。这些微塑料颗粒在河流、海洋等水体中广泛存在，被生物体摄入后，表现出多种生物学毒性，对水生生物构成了潜在的威胁。
目前对微塑料在水生生物体内的富集、分布有一定的研究进展，主要集中在使用显微镜对微塑料颗粒计数，从而大致判断微塑料在生物体不同器官和组织的分布情况。但是由于微塑料颗粒与其他类似环境颗粒在视野下难以分辨，需要借助傅里叶红外变换光谱仪、拉曼表征等对颗粒进行定性表征，因此增大了微塑料在生物体内定位与定量的工作量与复杂度。且通过计数方式得到的数据存在主观性与取样的误差，无法对微塑料的含量进行准确的定量。此外，现有的其他定量技术需要复杂的预处理过程(如消解生物组织)，进而提取出组织内的微塑料颗粒，增大了检测的工作量，因此开发一种无需复杂预处理、基于荧光活体成像的方法，用于研究微塑料在活体内的分布、定量具有重要的环境意义。
发明内容
针对现有技术预处理过程复杂、且无法对水生生物活体内的微塑料分布与 含量进行分析的局限，本发明提供了一种分析海洋刺胞生物中微塑料含量与分布的方法，包括：
暴露实验：选择特定直径的荧光微塑料颗粒加入到人工海水中配置成暴露溶液体系，选择海洋刺胞生物作为受试对象开展暴露实验。
体式荧光显微镜观察：将暴露后的受试对象活体或局部组织转移至体式荧光显微镜下，利用体式荧光显微镜观察微塑料在整只受试对象或局部组织中的分布。
样本荧光成像与微塑料含量计算：将暴露后的受试对象活体或局部组织置于小动物活体成像仪检测室中，采用小动物活体成像仪测定受试对象活体或局部组织中的荧光强度，并根据标准曲线计算相应含量；其中，所述荧光微塑料标准曲线通过如下方法绘制获得：
将盛有不同含量荧光微塑料的溶液滴加到培养皿中，放置于小动物活体成像仪检测室中，设置特定激发波长和发射波长后测定各溶液的荧光强度，以微塑料含量与对应的荧光强度绘制出荧光微塑料标准曲线。
进一步地，受试对象是海洋刺胞生物，为水母或海葵等。
进一步地，所述暴露溶液体系中，人工海水盐度为20‰-30‰；暴露溶液温度为18-25℃；微塑料直径为100-1000nm。
进一步地，利用培养皿作为容器将暴露后的受试对象活体或局部组织转移至体式荧光显微镜下和小动物活体成像仪检测室中。
优选地，荧光显微镜滤光片为GFP-B；
进一步地，所述暴露实验具体为：将受试对象置于暴露溶液体系中培养一段时间后，捞出，用人工海水清洗后待检测。
进一步地，所述暴露实验还包括设置空白对照组，用于样本荧光成像与微 塑料含量计算中扣除受试对象本底荧光值，所述空白对照组的海洋刺胞生物在人工海水中培养。
进一步地，使用小动物活体成像仪对受试对象活体进行荧光成像。
进一步地，还包括：将检测后的受试对象冷冻干燥并称量样品干重，结合样品荧光成像得到的微塑料含量计算得到单位质量组织中微塑料的含量。
进一步地，冷冻干燥时间为48小时。
本发明具有如下优点：本发明方法直接采用体式荧光显微镜、小动物活体成像仪对海洋刺胞生物进行微塑料检测，无需消解生物组织等处理过程，不仅能准确定位微塑料在活体海洋刺胞生物体内的分布，而且极大地简化了检测过程，并有效对海洋刺胞生物体内的微塑料含量进行准确定量，对新型环境污染物的监测与治理具有重要意义，为研究微塑料在海洋刺胞生物体内的富集能力提供了方法。
附图说明
图1为本发明方法的流程图。
图2荧光微塑料在水母全身的分布图。
图3荧光微塑料在水母口腕和伞体中的分布图。
图4荧光微塑料的荧光强度随含量变化的标准曲线图。
图5在不同浓度荧光微塑料的海水暴露下水母荧光活体成像图。
图6荧光微塑料在水母体内的含量。
具体实施方式
本发明公开了一种分析海洋刺胞生物中微塑料含量与分布的方法，为微塑料健康风险评价奠定基础。该方法的流程如图1所示，包括以下步骤：
(1)海洋刺胞生物暴露实验：选择特定直径的荧光微塑料颗粒加入到人工海水中配置成暴露溶液体系，选择海洋刺胞生物作为受试对象开展暴露实验；
(2)体式荧光显微镜观察：将暴露后的受试对象转移到培养皿上，再将培养皿置于体式荧光显微镜下，利用体式荧光显微镜观察微塑料在整只海洋刺胞生物活体中的分布。也可以用解剖工具将受试对象分成各组织部位后，依次观察，定位微塑料具体位置；
(3)样本荧光成像与微塑料含量计算：将暴露后的受试对象转移到培养皿上，将培养皿置于小动物活体成像仪检测室中，采用小动物活体成像仪测定整只海洋刺胞生物活体中荧光强度，并根据荧光微塑料标准曲线计算相应的微塑料含量；同样地，该步骤中，也可以用解剖工具将海洋刺胞生物分成各组织部位后转移到培养皿上，将培养皿置于小动物活体成像仪检测室中，采用小动物活体成像仪测定各组织中的荧光强度，进而根据荧光微塑料标准曲线计算相应的微塑料含量；
其中，荧光微塑料标准曲线绘制方法为：将盛有不同含量荧光微塑料的溶液滴加到培养皿中，放置于小动物活体成像仪检测室中，设置特定激发波长和发射波长(依据选择的荧光标记物设置)后测定各溶液的荧光强度，以微塑料含量与对应的荧光强度绘制出标准曲线；
(4)样本冻干与微塑料浓度计算：将检测后的样品置于冷冻干燥仪中干燥并称量样品干重，结合步骤(3)的微塑料含量换算得到单位质量组织中微塑料的含量。
在上述步骤中优选地是，所述步骤(1)使用的是荧光标记的微塑料颗粒，直径在20-1000nm；
在上述方案中优选的是，所述步骤(1)使用的受试对象是水母，如海月水 母、海蜇等；
以下以海蜇幼体为例对本发明进行详细说明。实施实例只用于对本发明进一步说明，不代表本发明的保护范围，其他人根据本发明做出的非本质的修改和调整，依然属于本发明的保护范围。
实施例
一种分析海洋刺胞生物中微塑料含量与分布的方法，如图1所示，包括以下步骤：
(1)水母暴露实验：以NBD-CL(激发波长488nm，发射波长518nm)荧光染料标记的聚苯乙烯微球(PS-MP，直径1μm)作为受试物，加入到人工海水中，配置成暴露溶液。受试动物为健康的海蜇幼体(伞径2.5±0.5cm)，随机分为暴露组和对照组。暴露组包括3个聚苯乙烯微球浓度水平(0.1,1,10mg L-1)，暴露周期为5d。其他实验条件如下：温度：22±1℃，盐度：25±1‰，光照周期：12h光照，12h黑暗交替，每天喂食丰年虾无节幼体。将暴露组活体水母置于盛有暴露溶液的容器，对照组活体水母置于人工海水中培养一段时间后，用不锈钢药匙将水母从玻璃瓶中捞出，用干净的海水清洗水母表面的微塑料，再转移到干净的海水中，以上步骤重复三次。
(2)体式荧光显微镜观察：水母经荧光微塑料暴露5d后(暴露浓度：10mg L-1)，使用体式荧光显微镜(Nikon SMZ18，日本)定位微塑料在整只活体水母中的分布。体式荧光显微镜的滤光片为GFP-B(参数：EX 480/40、DM 505、BA 535/50)。如图2所示，可以清晰观察到整只水母活体中荧光微塑料的分布情况，不同组织部位，微塑料富集的分布不同。之后，为了更好地确定微塑料在水母体内的组织分布，用解剖工具将水母分为伞体和口腕，再用体式荧光显微镜观察，结果如图3所示。
(3)荧光微塑料标准曲线绘制：配制不同微塑料含量的梯度荧光微塑料溶液，吸取1mL标准溶液滴到培养皿中，放置在小动物活体成像仪(IVIS Spectrum公司，美国)检测室中，设置特定激发波长和发射波长后测定各溶液的荧光强度，绘制荧光强度随微塑料含量变化的标准曲线；本实施例中荧光微塑料的标准曲线设置微塑料含量为0,5,20,50,100,200μg，标准曲线如图4所示。
(4)样本荧光成像与微塑料含量计算：将活体水母转移到培养皿上，在步骤(3)的激发和发射波长下，采用小动物活体成像仪测定水母组织中荧光强度，并根据标准曲线计算相应含量；其中暴露组样品的结果要扣除空白对照组的背景值，得到最终结果；图5所示为不同浓度荧光微塑料海水暴露下水母的荧光活体成像图。
(5)样本冻干与微塑料浓度计算：将检测后的样品置于冷冻干燥仪中干燥48h后称重，得到水母组织干重，结合步骤(4)的微塑料含量换算得到单位质量组织中荧光微塑料的含量，如图6所示。
本发明方法直接采用体式荧光显微镜、小动物活体成像仪对海洋刺胞生物进行微塑料检测，无需消解生物组织等处理过程，不仅能准确定位微塑料在活体海洋刺胞生物体内的分布，而且极大地简化了检测过程，并有效对海洋刺胞生物内的微塑料含量进行准确定量，对新型环境污染物的监测与治理具有重要意义。

Claims (7)

  1. 一种分析海洋刺胞生物中微塑料含量与分布的方法，其特征在于，所述方法包括：
    选择特定直径的荧光微塑料颗粒加入到人工海水中配置成暴露溶液体系，将海洋刺胞生物作为受试对象开展暴露实验；
    将暴露后的受试对象活体或局部组织转移至体式荧光显微镜下，利用体式荧光显微镜观察微塑料在整只受试对象或局部组织中的分布；将暴露后的受试对象活体或局部组织置于小动物活体成像仪检测室中，采用小动物活体成像仪测定受试对象活体或局部组织中的荧光强度，并根据荧光微塑料标准曲线计算微塑料含量；
    其中，所述荧光微塑料标准曲线通过如下方法绘制获得：
    将盛有不同含量荧光微塑料的溶液滴加到培养皿中，放置于小动物活体成像仪检测室中，设置特定激发波长和发射波长后测定各溶液的荧光强度，以微塑料含量与对应的荧光强度绘制出荧光微塑料标准曲线。
  2. 如权利要求1所述的方法，其特征在于，所述海洋刺胞生物为水母或海葵。
  3. 如权利要求1所述的方法，其特征在于，所述暴露溶液体系中，人工海水盐度为20‰-30‰；暴露溶液温度为18-25℃；微塑料直径为100-1000nm。
  4. 如权利要求1所述的方法，其特征在于，所述暴露实验具体为：将受试对象置于暴露溶液体系中培养一段时间后，捞出，用人工海水清洗后待检测。
  5. 如权利要求1所述的方法，其特征在于，所述体式荧光显微镜的滤光片为GFP-B。
  6. 如权利要求1所述的方法，其特征在于，还包括：将检测后的受试对象冷冻干燥并称量样品干重，结合样品荧光成像得到的微塑料含量计算得到单位质量 组织中微塑料的含量。
  7. 如权利要求6所述的方法，其特征在于，冷冻干燥的时间为48小时。
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